Photochemistry and Photobiology Vol. 44, No. 3 , pp. 355 - 358, 1986 0031-8655186 $03.

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Printed in Great Britain Peigamon Journals Ltd

ABSORPTION SPECTRA OF DEOXYRIBOSE,

RIBOSEPHOSPHATE, ATP AND DNA BY DIRECT
TRANSMISSION MEASUREMENTS IN THE VACUUM-UV
(150-190 nm) AND FAR-UV (190-260 nm) REGIONS USING
SYNCHROTRON RADIATION AS A LIGHT SOURCE
ITOand TAKASHI
ATSUSHI ITO*
Institute of Physics, College of Arts and Sciences, University of Tokyo, Meguroku, Komaba 3-8-1,
Tokyo 153, Japan

(Received 8 January 1986; accepted 13 January 1986)

Abstract-Transmission measurements of 2-deoxy-~-ribose,~-ribose-5-phosphate,ATP and DNA at 5

nm intervals were made with thin films in the wavelength region between 150 nm and 260 nm using
synchrotron radiation. ATP and DNA exhibited two peaks in the absorption spectra around 260 nm and
190 nm, and a steep increase below 170 nm, while ribose phosphate and deoxyribose only exhibited the
increase below 190 nm with no appreciable absorption above 190 nm. Since adenine does not exhibit the
increase of absorption below 180 nm, these results indicate that the absorption of the sugar-phosphate
group, rather than adenine, contributed to the increase below 170 nm in the absorption spectra of ATP
and DNA.

INTRODUCTION ATP, and DNA in the wavelength range between 150

nm and 260 nm at 5 nm intervals. By using SR we
The absorption spectrum of DNA has been mea- could select any wavelength, not only the discrete
sured by Inagaki et d.(1974) in the spectral range of spectral lines usually available with conventional
2-82 eV (62C-15 nm) and by Sontag and Weibezahn light sources. Special attention was taken to use
(1975) from .3-25 eV (415-50 nm). Both studies uniform films of ATP of various thicknesses in order
demonstrated the existence of a stronger absorption to determine the absorption cross-section in absolute
band around 190 nm than the well-known peak at units (m2). In the cases of ribosephosphate and
260 nm. Below 180 nm a steep increase has com- deoxyribose, the films were prepared from solutions
monly been observed. Yamada and Fukutome of the same molar concentration so that we could
(1968) have shown the same 190 nm peak with make a direct comparison between the two spectra.
nucleic acid bases with nearly flat absorption down
to 120 nm. Onari (1969) measured the absorption MATERIALS AND METHODS
spectra of synthetic polynucleotides and ribose Materials. ATP (5’-adenosine triphosphate, disodium
phosphate; he regarded the spectra of polynuc- salt) was purchased from Seikagaku Kogyo Co. and used
leotides above 6 eV (below 207 mm) as the super- without further purification. Polyvinylalcohol (PVA),
position of the absorption of bases and ribose phos- which usually was added to the sample solution for a better
uniformity in thickness, was purchased from Tokyo Kasei
phate. Kiseleva et aZ. (1975) made more systematic Co. ~-Ribose-5-phosphateand 2-deoxy-D-ribose were
measurements with DNA, RNA, nucleotides, nuc- purchased from Sigma Chemical and Wako Pure Chemical
leosides, sugar and phosphate. They assigned the Industries, LTD, respectively. DNA (highly polymerized)
increase of absorption in nucleosides, starting from was obtained from Nutritional Biochemical Co.
160 nm and continuing toward the shorter Preparation ofthinfilms. A polished CaF, plate (1.5 mm
thick, 20 mm 4, 70% transmittance at 150 nm, Oyokoken
wavelengths, as the absorption of sugar and phos- Co.) provided a substrate for the samples.
phate. The fact that the same increase was seen in The same procedures were used for the preparation of the
the spectra of both sugar and phosphate was consi- thin films of ribose phosphate and deoxyribose. A mixture
dered as supporting this assignment. However, in- of 0.1 me consisting of equal amount of ribosephosphate or
deoxyribose (0.1 M) and PVA (1.75 mgime) was dropped
sufficient details have been presented, especially in onto the CaF, plate and left to dry in air; drying started from
vacuum-ultraviolet (vacuum-UV) region, to enable the center and yielded a uniform thin film especially in the
us to evaluate those spectra critically. center.
Using synchrotron radiation (SR)t as a light In the case of ATP, thin films with different thicknesses
were achieved by systematically changing the concentration
source, we have made detailed transmission of ATP in the range from 2 X to 1 x lO-’M, while that
measurements of deoxyribose, ribosephosphate, of PVA was kept constant at 1.75 mgime.
DNA films were prepared by dropping 0.2 me DNA
solution (1.5 mglme) on the CaF, plate. As the DNA
*To whom correspondence should be addressed. solution was highly viscous at this concentration, no addi-
?Abbreviations: Vacuum-UV, vacuum-ultraviolet; SR, tion of PVA was necessary to obtain a uniform film.
synchrotron radiation; ATP, 5’-adenosine triphosphate; The uniformity of the sample films was ascertained to be
PVA, polyvinylalcohol. within several percent over the central area of the sample on
355
356 ITOand TAKASHI
ATSUSHI ITO

the CaF, plate by absorption measurements at 260 nm, conventional spectrophotometer. Clearly, the data
using a chromatoscanner (CS-930, Shimazu Co.) with the points at 160 and at 190 nm were linear. This finding
beam area set as 0.2 x 3 mm. The SR beam size used for the assured the proper choice of the thickness range for
transmission measurement was smaller (6 x 3 mm) than the
area of the film that we ascertained to be uniform. determination of the absorption coefficient. Assum-
Transmission measurements. The measurements were ing that linearity of absorbance with thickness holds
made using a recently completed monochromatic SR system for other wavelengths, we were able to calculate the
(Ito el al., 1984) installed at BL-5 port of the INS-SOR absorption cross-section,
storage ring (Synchrotron Radiation Laboratory, Institute
of Solid State Physics, University of Tokyo). The CaF, plate The absorption cross-section u (cm2) is defined by
loaded with the sample was set in one of the six positions of the Eq 1,
the sample holder with the reference sample (either a plain
CaF2 plate or a CaF, plate loaded with solvent materials) at
another position. A nearly parallel beam of SR (6 mm long,
3 mm wide) passed through the sample film and the backing
CaF, plate, then impinged on a sodium salicylate-coated flat where I, = intensity of incident radiation
glass, where incoming ultraviolet radiation was converted to I = intensity of transmitted radiation
visible light that was detected by a photomultiplier (R-268, c = concentration of sample (molecules/
Hamamatsu Photonics Co.). The bandwidth of the incom- cm3)
ing radiation on the sample area was 5 nm. The photomulti- d = thickness of sample (cm).
plier output was fed into a computer-operated data acquisi-
tion system for processing to yield the final output normal- Since cd = (N/V)d = N/S, Eq. 1may be expressed as
ized at unit ring current of the storage ring. This output was
used for calculation of the absorbance A according to Eq. 3 -!. = e-u(NIS)
(2)
in Results and Discussion. 1,
where Nis the number of molecules present in the
RESULTS AND DISCUSSION volume V , of the sample defined by the
beam area, S.
The absorption spectra of ATP, expressed in On the other hand, the absorbance, A , may be
absorbance units for three different thicknesses of expressed as
film are shown in Fig. 1. All spectra have the same
I
- = 10-A =
profile with two maxima: one is around 260 nm (data e-A loge 10
(3)
above 260 nm are not given) which is the well-known 10
peak in the far-UV region, and there is another By comparing Eq. 1 with Eq. 3, we obtain
broader one around 190 nm. From 180 nm to the
shorter wavelengths there is a sharp increase of u = A (log, 10) (SIN) (4)
absorption. The figure insert shows the change in where A and S are either measurable or known
absorbances as a function of thickness of the sample parameters. Under a given condition, N was deter-
films at 160and 190nm in reference to that of 260 nm, mined photometrically at 260 nm by dissolving a 6 x 3
where a precise measurement was carried out with a mm area of the film. By substituting the values of A,

7 -, 0.6
-
N
a5
OA
nm

9 nm
X
h
2 a3 - 0.5
N 0
E
v Q2 nm
6
5
L ~ -
C u)
0.1 - 0.4
.-
c
0 Q,

U 0 2 U
0,
m o 0.1 a2 0.3 0.4 C
I
m Thickness of m e film 0.3
In (in absorbance units at 260 0
2
0
D
nm 1 10.2
-
L
C
.-0
\
0

P
0
In
m 0.1 n
n
a a
0
150 200 2 50 300
W a v e l e n g t h (nm)
Figure 1. Absorption spectra of ATP with varying film thicknesses. The insert show the relationship
between absorbance and sample thickness at 160, 190, and 260 nm (see text for details).
 Absorption spectra of ATP and related compounds in VUV region 357

Table 1. Absorption cross-section of ATP O* [ 2-Wy-D-Rlbou

Wavelength Absorption cross-section a2

(nm) (m*/photon) x loz1
- I

150 6.99
155 6.48
160 5.37
165 4.76
170 4.44
175 4.22
180 3.62
185 3.46
I
190 3.76
195 3.71 150 Mo 250 300
200 3.69 Wovrlcngth (nm)
205 3.50
210 3.25 Figure 2. Absorption spectra of 2-deoxy-~-ribose and
215 3.02 D-ribose 5-phosphate film.
220 2.38
225 1.78
230 1.22
235 0.96 0.6 r
240 0.99
245 1.14
250 1.44
255 1.76 0
260 1.91 u
E
0
n
L
N and S (= 6 x 3 mm) into Eq. 4, u was calculated 0
Ln
(Table 1). Figure 1 also shows the u-spectrum in n
4
absolute units using one of the absorbance spectra
mentioned above.
Kiseleva et al. (1975) have reported a similar
spectrum of ATP with two peaks around 260 nm and 150 200 250 300
200 nm, a deep minimum around 160 nm and a steep Wavelength (om)
increase below 150 nm. To compare our results with
Figure 3. Absorption spectrum of DNA film.
theirs in more detail, relative absorptions normalized
at 260 nm are listed in Table 2a. The large discrepan-
cies below 160 nm may be attributable to the general
shift of their spectrum toward the shorter (Yamada and Fukutome, 1968). The increase of
wavelengths. However considering the fact that a absorption below 180 nm may be assigned to the
thicker film of deoxyadenosine exhibited a deeper sugar-phosphate group, since both ATP and
minimum at 160 nm (Kiseleva et al., 1975), these ribosephosphate showed a similar structure, whereas
differences might result from the effects of film adenine alone did not show such an increase (Yama-
thickness. We believe that thinner film is better to da and Fukutome, 1968). These assignments are also
avoid effects such as aggregation and stacking. The supported by another report in which it was shown
linearity check that we performed may be an impor- that the absorption of deoxyribose and orthophos-
tant consideration in determining the absorption phate (Na3P04)began below 200 nm (Kiseleva et al.,
when film samples are used. 1975).
Figure 2 shows the absorption spectra of 2-deoxy- The absorption spectrum of a DNA film on CaF2,
D-ribose and ~4bose-5-phosphate.Since the same measured in the same manner as above, is presented
molar concentration was used in making up the films in Fig. 3. The spectrum had two peaks as in ATP, and
on the CaF2 plate, the films should have approx- showed a sharp increase below 170 nm. The relative
imately the same number of molecules. In both absorption is listed in Table 2b together with figures
spectra the onset of absorption was seen at 190 nm, calculated from data given by Inagaki et al. (1974)
although the increase of absorption by ribosephos- and Sontag and Weibezahn (1975). Athough our
phate was much steeper than by ribose alone. figures are somewhat higher at shorter wavelengths
From the absorption spectra of ATP, deoxyribose than theirs, a general agreement can be seen. The
and ribosephosphate described, we conclude that the spectral data of Inagaki et al. (1974) were based on
two peaks at 260 nm and 190 nm in ATP are due to the determination of two optical constants, the
the adenine base because these peaks were not seen dimensionless absorption coefficient k and the refrac-
in the spectra of deoxyribose and ribosephosphate tive index n . They actually measured the transmis-
but occurred in the spectrum of adenine alone sion of a free standing DNA film with a fixed
358 ATSUSHIITO and TAKASHI
ITO

Table 2. Comparisons of absorption of ATP and DNA film at selected

wavelengths

Wavelength (nm)

260* 190 160 150

(a) ATP
This study 1 1.97 2.81 3.66
Kiseleva et al. 1 1.13 0.67 0.77
(b) DNA
This study 1 1.86 1.98 2.18
Inagaki et al. 1 1.23 1.18 1.36
Sontag and Weibezahn 1 1.42 1.50 1.91

*All data were normalized to 1 at 260 nm.

Kiseleva et al. (1975) employed essentially the same transmission measure-
ments as the present study. Inagaki et al. (1974) determined optical constants,
dimensionless extinction coefficient k and refractive index n. We converted k
into the more common extinction coefficient with units of cm-' by multiplying
k by 4 K ' . Sontag and Weibezahn (1975) measured (below 200 nm)
scattered photons by converting them to electrons on a CsJ coated spherical
cathode with the sample film on the center.

thickness and determined k by taking account of the REFERENCES

refractive index It. We have converted k to the more Inagaki, T., R. N. Hamm and E. T. Arakawa (1974)
common extinction coefficient with the units cm-' to Optical and dielectric properties of DNA in me extreme
ultraviolet. J . Chem. Phys. 61, 4246-4250.
compare with our spectrum. Sontag and Weibezahn Ito, T., T. Kada, S . Okada, K. Hieda, K. Kobayashi, H.
(1975) based their spectrum of free-standing DNA Maezawa and A. Ito (1984) Synchrotron system for
film on the measurements of scattered photons by monochromatic UV irradiation (> 140 nm) of biological
converting them to electrons on a CsJ coated- material. Radiat. Res. 98, 65-73.
Kiseleva, M. N., Ye. P. Zarochentseva and N. Ya.
spherical cathode. Dodonova (1975) Absorption spectra of nucleic acids
and related compounds in the spectral region 120-280
nm. Biophysics (Biofizika) 20, 571-575.
Onari, S. (1969) Vacuum ultraviolet absorption spectra
Acknowledgements-We acknowledge the use of synchrot- of homo-polynucleotides. J . Phys. SOC. Japan 26, 214.
ron radiation facilities at the Synchrotron Radiation Sontag, W. and K. F. Weibezahn (1975) Absorption of
Laboratory, University of Tokyo. Also we thank Drs. DNA in the region of vacuum-UV (3-25 eV). Rad.
Hieda, H. Maezawa and other members of the cooperative Environ. Biophys. 12, 169-174.
project for assisting the experiments. This work was sup- Yamada, T. and H. Fukutame (1968) Vacuum ultra-
ported by a Grant-in-Aid from the Ministry of Education, violet absorption spectra of sublimed films of nucleic acid
Science and Culture, Japan. bases. Biopolymers 6, 43-54.